IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/09/12

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(RBS screening (single digest))
Current revision (05:46, 16 September 2008) (view source)
(RBS screening (single digest))
 
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- S11 with XbaI : uncut, self ligation
- S11 with XbaI : uncut, self ligation
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==Double check of RBS S6 and stock==
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- Grow RBS S6 in LB with Cm35
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==Check more RBS W==
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- Grow RBS W2, 4, 6, 8, 10 in 10mL of LB with antibiotic
==Check PCR products==
==Check PCR products==

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RBS screening (single digest)

- Plasmid miniprep 6 colonies of RBS S and 6 colonies of RBS W (without endo wash buffer)

- Nanodrop

product concentration (ng/μL) 260/280 quantity of DNA to add to have about 300ng (μL) added SDW for single digest (μL)
RBS W1 57.1 1.57 6 11
RBS W3 54.1 1.53 6 11
RBS W5 15.1 1.85 17 0
RBS W 7 21.8 1.66 15 2
RBS W9 21.8 1.78 15 2
RBS W11 13.8 1.73 17 0
RBS S2 19.5 1.7 15 2
RBS S4 82.13 2.57 4 13
RBS S6 44.7 1.64 7 10
RBS S8 104.0 1.48 3 14
RBS S10 13.1 1.82 17 0
RBS S11 20.1 1.73 15 2

- Single digest : with EcoRI and XbaI, add SDW and DNA (according to the previous table, 300mg of DNA), 2μL of fast digest buffer, and 1μL of enzyme

- Gel1

  • Lane2 : HyperladderI
  • Lane3 : RBS W1 cut with EcoRI
  • Lane4 : RBS W1 cut with XbaI
  • Lane5 : RBS W3 cut with EcoRI
  • Lane6 : RBS W3 cut with XbaI
  • Lane7 : RBS W7 cut with EcoRI
  • Lane8 : RBS W7 cut with XbaI
  • Lane9 : RBS W9 cut with EcoRI
  • Lane10 : RBS W9 cut with XbaI
  • Lane11 : RBS W9 uncut
  • Lane12 : supercoiled ladder


- Gel2

  • Lane2 : HyperladderI
  • Lane3 : RBS S4 cut with EcoRI
  • Lane4 : RBS S4 cut with XbaI
  • Lane5 : RBS S6 cut with EcoRI
  • Lane6 : RBS S6 cut with XbaI
  • Lane7 : RBS S8 cut with EcoRI
  • Lane8 : RBS S8 cut with XbaI
  • Lane9 : RBS S11 cut with EcoRI
  • Lane10 : RBS S11 cut with XbaI
  • Lane11 : RBS S4 uncut
  • Lane12 : supercoiled ladder
  • Result

The size of our plasmid is about 3000bp (ok on the gel)

  • RBS W

- W1 with EcoRI : cut

- W1 with XbaI : uncut, self ligation

- W3 with EcoRI : cut

- W3 with XbaI : uncut, self ligation

- W7 with EcoRI : impossible to see

- W7 with XbaI : uncut, self ligation

- W9 with EcoRI : cut

- W9 with XbaI : uncut, self ligation

  • RBS S

- S4 with EcoRI : cut

- S4 with XbaI : uncut, self ligation

- S6 with EcoRI : cut

- S6 with XbaI : 2 band, this plasmid is partially cut!!! Transformation with RBS S

- S8 with EcoRI : cut

- S8 with XbaI : uncut, self ligation

- S11 with EcoRI : cut

- S11 with XbaI : uncut, self ligation

Double check of RBS S6 and stock

- Grow RBS S6 in LB with Cm35

Check more RBS W

- Grow RBS W2, 4, 6, 8, 10 in 10mL of LB with antibiotic

Check PCR products

- Gel (2μL of each product)

  • Lane1 : λ ladder
  • Lane2 : agrA
  • Lane3 : agrB
  • Lane4 : agrC
  • Lane5 : rep
  • Lane6 : pSB4C5 1
  • Lane7 : pSB4C5 2
  • Lane8 : Pxyl
  • Lane9 : Ppac
  • Lane10 : RBS S
  • Lane11 : RBS W
  • Lane12 : HyperladderI
  • Results

- agr B : 2 bands?

- agrC : ok

- rep : small band but good size

- backbones : ok

- promoters : ok

- RBS : nothing


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